Project Summary/Abstract Despite several new treatment options, many patients with rheumatoid arthritis (RA) continue to suffer from poorly controlled disease. To enable the urgently needed development of better therapies, the molecular mechanisms that underpin the pathogenesis of RA need to be better understood. The newest hypothesis postulates that abnormally citrullinated proteins become `neo'-self-antigens and stoke direct autoimmunity against this modified self. Key questions now are why and how excessive and/or abnormal citrullination occurs in RA patients. The research proposed here will provide novel insights into how pathological citrullination occurs in RA patients. This work will help elucidate the molecular mechanisms by which the citrullinating enzymes PAD2 and/or PAD4 act in triggering RA. This will, in turn, be invaluable for evaluating them as drug targets, and will provide biomarker opportunities to monitor the activity of the disease and possibly stratify RA patients into different subpopulations that may require different therapies. The Specific Aims are: AIM 1. The mechanism(s) of increased citrullination in RA patients. AIM 2. A novel function of PAD4 in oxidative burst regulation. The first Aim directly seeks evidence in patient samples of the five proposed mechanisms of pathological citrullination to determine which are operational in vivo. Their contributions individually, or in combinations, to protein citrullination of disease-relevant substrates, including substrate mixtures and cells relevant to RA pathogenesis will be investigated. The second Aim explores the discovery of a novel link between PAD4 and the cytosolic components of the NADPH oxidase machinery (NOX2) in human neutrophils. The citrullination of NOX2 components may contribute to the pathogenesis of RA by blocking a protective oxidative burst response. The molecular details of PAD4 interaction with NOX2 will be examined and the citrullination of specific arginine residues and the functional consequences of these sites on the oxidative burst response tested, as this response has been reported to be involved in tempering autoimmunity.